DNA polymerase iota (Pol iota) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol iota, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol iota, and knockin mice which express full length Pol iota that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol iota did not change overall mutagenesis. We then examined if Pol iota affected tandem mutations generated by another error prone polymerase, Pol zeta. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol iota-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol iota occasionally accesses the replication fork to generate a first mutation, and Pol zeta extends the mismatch with a second mutation.